The present invention is directed to improved methods and systems for preparing cDNA molecules, especially in the form of cDNA libraries. Preferred embodiments of the invention include methods and systems capable of generating cDNA libraries enriched for high molecular weight cDNA inserts.
With the various genome projects in the process of completion, the field of functional genomics is currently faced with the challenge of first identifying genes and then identifying the function of the different proteins encoded by these genes. The human genome is estimated to contain about 100,000 genes (Borrebaeck, C. A. (1998) Immunol. Today 19: 524-7). Therefore, the main focus in the forthcoming era will be on identification of the role of these genes in normal and disease states. This will consequently result in the design of more effective treatments. Novel, automated, high-capacity, and reliable technologies have to be developed to ensure the success of this complex task. This includes the invention and development of easier and faster methods of RNA isolation, purification, reverse transcription, cDNA library construction and screening, gene expression, translation, and protein purification, as well as mutation detection. The instant invention represents such a method.
In one aspect, the present invention concerns an improved method for the preparation of cDNA libraries. Preferred embodiments of the invention include methods and systems capable of generating cDNA libraries enriched for high molecular weight cDNA inserts.
In one embodiment, the invention provides a method for preparing a fraction of mRNA molecules suitable for use in the production of a cDNA library enriched for inserts of a desired relative size range comprising the steps of applying a plurality of mRNA molecules to a separation medium having a non-polar surface in the presence of a counterion agent, wherein said plurality of mRNA molecules comprises mRNA molecules of diverse sizes; eluting at least a portion of the plurality of mRNA molecules from the separation medium by means of a mobile phase that includes a solvent that is less polar than water, whereby the plurality of mRNA molecules is fractionated in a manner that is at least partially dependent upon mRNA size; and collecting a fraction of mRNA molecules as it elutes from the column, wherein the fraction of mRNA molecules collected is enriched for mRNA molecules of a desired size range relative to the plurality of mRNA molecules applied to the column, such that the fraction of mRNA molecules collected is suitable for use in the production of a cDNA library enriched for inserts of a desired relative size range.
In another embodiment, the invention provides a method for preparing a cDNA library enriched for inserts of a desired relative size range comprising the steps of: applying a plurality of mRNA molecules to a separation medium having a non-polar surface in the presence of a counterion agent, wherein said plurality of mRNA molecules comprises mRNA molecules of diverse sizes; eluting at least a portion of the plurality of mRNA molecules from the separation medium by applying a mobile phase that includes a solvent that is less polar than water, whereby the plurality of mRNA molecules is fractionated in a manner that is at least partially dependent upon mRNA size; collecting a fraction of mRNA molecules as it elutes from the column, wherein the fraction of mRNA molecules collected is enriched for mRNA molecules of a desired size range relative to the plurality of mRNA molecules applied to the column; and reverse transcribing the collected fraction of mRNA molecules to form a library of cDNA inserts enriched for inserts of a desired relative size range.
In a preferred embodiment of the invention, the fraction of mRNA molecules collected is enriched for the larger-size constituents of the plurality of mRNA molecules applied to the separation medium.
In another preferred embodiment, the library of cDNA inserts comprises cDNA inserts residing in nucleic acid vectors, such as plasmids and phage vectors, which are preferably maintained in host cells.
In another preferred embodiment, the plurality of mRNA molecules applied to the separation medium comprises a sample of total RNA or total mRNA from a biological sample.
In yet another preferred embodiment, the separation of mRNA molecules is achieved by Ion Pairing Reversed Phase HPLC. Alternatively, the separation of mRNA molecules can be achieved in a batch process, for example by use of an apparatus selected from the group consisting of spin columns, vacuum tray devices low pressure columns and medium pressure columns.
In a preferred embodiment of the invention, the separation of mRNA molecules is achieved under denaturing conditions. mRNA denaturation can be achieved by conducting the separation at a temperature sufficient to denature at least some portion of the plurality of mRNA molecules applied to the separation medium, by conducting the separation in the presence of a chemical denaturant, or by conducting the separation at a pH sufficient to denature at least some portion of the plurality of mRNA molecules applied to the separation medium.
Preferably the separation of mRNA molecules is conducted at a temperature greater than about 50xc2x0 C., more preferably at a temperature of about 75xc2x0 C. or greater.
The separation is preferably conducted under conditions that are substantially free of multivalent cations capable of interfering with polynucleotide separations.
In a preferred embodiment of the invention, the separation medium comprises particles selected from the group consisting of silica, silica carbide, silica nitrite, titanium oxide, aluminum oxide, zirconium oxide, carbon, insoluble polysaccharide, and diatomaceous earth, the particles having separation surfaces which are coated with a hydrocarbon or non-polar hydrocarbon substituted polymer, or have substantially all polar groups reacted with a non-polar hydrocarbon or substituted hydrocarbon group, wherein the surfaces are non-polar.
In another embodiment, the separation medium comprises polymer beads having an average diameter of 0.5 to 100 microns, the beads being unsubstituted polymer beads or polymer beads substituted with a moiety selected from the group consisting of hydrocarbon having from one to 1,000,000 carbons. A particularly preferred separation medium comprises C-18 alkylated nonporous poly(styrene-divinylbenzene) polymer beads.
In another embodiment, the separation medium comprises a monolith.
In a preferred embodiment of the invention, the separation medium is substantially free of multivalent cations capable of interfering with polynucleotide separations. The separation medium is preferably prepared using reagents that are substantially free of multivalent cations capable of interfering with polynucleotide separations and under conditions that are substantially free of multivalent cations capable of interfering with polynucleotide separations.
In a preferred embodiment of the invention, the separation medium has been subjected to acid wash treatment to remove any residual surface metal contaminants and/or subjected to treatment with a multivalent cation-binding agent.
In a preferred embodiment of the invention, the mobile phase includes an organic solvent selected from the group consisting of alcohol, nitrile, dimethylformamide, tetrahydrofuran, ester, ether, and mixtures of one or more thereof, where acetonitrile is particularly preferred.
In another preferred embodiment of the invention, the mobile phase includes a counterion agent selected from the group consisting of lower alkyl primary amine, lower alkyl secondary amine, lower alkyl tertiary amine, lower trialkylammonium salt, quaternary ammonium salt, and mixtures of one or more thereof. Particularly preferred is the use of a counterion agent selected from the group consisting of octylammonium acetate, octadimethylammonium acetate, decylammonium acetate, octadecylammonium acetate, pyridiniumammonium acetate, cyclohexylammonium acetate, diethylammonium acetate, propylethylammonium acetate, propyidiethylammonium acetate, butylethylammonium acetate, methylhexylammonium acetate, tetramethylammonium acetate, tetraethylammonium acetate, tetrapropylammonium acetate, dimethydiethylammonium acetate, triethylammonium acetate, tripropylammonium acetate, tributylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, triethylammonium hexafluoroisopropyl alcohol, and mixtures of one or more thereof, of which tetrabutylammonium bromide and triethylammonium acetate are especially preferred.